Advances in DNA methylation and its role in cytoplasmic male sterility in higher plants

The impact of epigenetic modifications like DNA methylation on plant phenotypes has expanded the possibilities for crop development.DNA methylation plays a part in the regulation of both the chromatin structure and gene expression,and the enzyme involved,DNA methyltransferase,executes the methylation process within the plant genome.By regulating crucial biological pathways,epigenetic changes actively contribute to the creation of the phenotype.Therefore,epigenome editing may assist in overcoming some of the drawbacks of genome editing,which can have minor off-target consequences and merely facilitate the loss of a gene’s function.These drawbacks include gene knockout,which can have such off-target effects.This review provides examples of several molecular characteristics of DNA methylation,as well as some plant physiological processes that are impacted by these epigenetic changes in the plants.We also discuss how DNA alterations might be used to improve crops and meet the demands of sustainable and environmentally-friendly farming.

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

Background Understanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources.However,only a few cytoplasmic male sterility(CMS)lines of cotton have been produced due to several challenges,like inadequate variation of agronomic traits,incomplete sterility,weak resilience of restorer lines,and difficulty in combining strong dominance.Therefore,the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.Results Two F_(2) segregating populations of cotton were constructed from cytoplasmic male sterile lines(HaA and 01A,maternal)and restorer lines(HaR and 26R,paternal).Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants.Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants.However,sterile plants exhibited smaller floral organs,shortened filament lengths,and anther atrophy on the flowering day in comparison with the fertile plants.When performed scanning electron microscopy(SEM),the two F_(2) populations revealed morphological variations in the anther epidermis.Cellular analysis showed no significant differences in pollen development before pollen maturation.Interestingly,between the pollen maturation and flowering stages,the tapetum layer of sterile plants degenerated prematurely,resulting in abnormal pollen grains and gradual pollen degradation.Conclusion The results of this study suggest that fertility-restoring genes are controlled by a single dominant gene.Sterile plants exhibit distinctive floral morphology,which is characterized by stamen atrophy and abnormal anthers.Pollen abortion occurs between pollen maturity and flowering,indicating that premature tapetum degradation may be the primary cause of pollen abortion.Overall,our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations,mechanisms of sterility,and the role of sterile and restorer genes.

Influence of Soil Moisture and Air Temperature on the Stability of Cytoplasmic Male Sterility (CMS) in Maize (Zea mays L.)

Cytoplasmic male sterility (CMS) is a maternally inherited trait that suppresses the production of viable pollen. CMS is a useful biological tool for confinement strategies to facilitate coexistence of genetically modified (GM) and non-GM crops in case where it is required. The trait is reversible and can be restored to fertility in the presence of nuclear restorer genes (Rf genes) and by environmental impacts. The aim of this study was to investigate the influence of the level of irrigation on the stability of CMS maize hybrids under defined greenhouse conditions. Additionally the combination of irrigation and air temperature was studied. Three CMS maize hybrids were grown with different levels of irrigation and in different temperature regimes. Tassel characteristics, pollen production and fertility were assessed. The CMS stability was high in hot air temperatures and decreased in lower temperatures. The level of irrigation had no major effect on the level of sterility. The extent of these phenomena was depending on the genotype of CMS maize and should be known before using CMS for coexistence purposes.

RAPD and ISSR Markers of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat

LK783 was found to be a good fertility restorer for K-type male sterility of wheat (Triticum aestivum L.). RAPD and ISSR (inter-simple sequence repeat polymorphism) markers were employed to map the major restoring gene in LK783. Maintainer and restorer DNA pools were established using the extreme sterile and fertile plants among KJ5418A//911289/LK783 F 1 population, respectively. Four hundred and eighteen RAPD primers and 33 ISSR primers were used for screening polymorphisms between the two pools, and amplification bands using a RAPD primer of OPK18 and an ISSR primer of UBC-845 were found polymorphic between the two pools. Linkage analysis showed that OPK18 450 and UBC-845 800 were linked to the restoring gene in LK783. The distance between the restoring gene and OPK18 450 was (15.07±6.28) cM (centiMorgan), with the distance between the restoring gene and UBC-845 800 being (8.20±4.85) cM. The marker of UBC-845 800 was located on chromosome 1BS by amplifying nulli-tetrasomics and 1B ditelosomics of Chinese Spring with the primer of UBC-845, indicating that the restoring gene in LK783 was located on 1BS. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility of wheat would be facilitated by using the two markers.

Microtubule Structure and Male Sterility in a Gene-Cytoplasmic Male Sterile Line of Rice, Zhen Shan 97A

Histological changes that occur during microsporogenesis are documented in a gene-cytoplasmic male sterile rice ( Oryza saliva L.) line, Zhen Shan 97A, its maintainer line, Zhen Shan 97B, and the restorer line, Ce64 of a Mine hybrid rice production system. In the restorer line, Ce64, the developing microsporocytes have dense cytoplasm and a distinct set of circumferential microtubules around the nucleus. Successive cytokinesis results in the formation of tetrads. The microtubules within the cells of tetrads and microspores radiate from the surface of the nucleus towards the outer edge of the cytoplasm. Subsequent pollen development is normal. During the course of microspore formation tubulin speckles can be found in the cytoplasm. The general pattern of development and microtubule organization in the maintainer lined Zhen Shan 97B, is similar to Ce64, except that a few more tubulin speckles appear during microspore formation. In the case of the mate sterile line, Zhen Shan 97A, a number of abnormalities can be discerned during early microsporogenesis. These include vacuoles forming within the developing microsporocyte and faintly stained microtubules with no defined distribution pattern. Prominent tubulin speckles are common within the cytoplasm. For those microsporocytes that undergo meiosis, no defined organizational patterns of microtubules can be found within the tetrad. All microspores abort soon after. Abnormalities and defects in microtubule organization observed in Zhen Shan 97A showed that complex interactions between the cytoplasm and the nucleus began at very early stage of microsporocyte development.

Mapping of Fertility Restoring Gene for Aegilops kotschyi Cytoplasmic Male Sterility in Wheat Using SSR Markers

LK783 was found to be a good fertility restorer for K-type male sterility of wheat. Microsatel-lite markers were employed to map the major restoring gene in LK783. Maintainer and restorer DNA pools were established using the extreme sterile and fertile plants among (KJ5418A//911289/LK783)F1 population, respectively. Seventy-nine sets of SSR primers were screened for polymorphism between the two pools, 6 of which were found polymorphic. Linkage analysis showed that Xgwm11, Xgwm18, Xgwm264a and Xgwm273 were linked to the restoring gene in LK783, while Xgwm11, Xgwm18 and Xgwm273 were co-segregated. The distance between the Rf gene in LK783 and the three co-segregated markers was 6.54 ± 4.37 cM, the distance between Rf gene and Xgwm264a was 5. 71 ± 4.10 cM. The four SSR markers were located on chromosome IBS by amplifying the DNA of nulli-tetrasomics and ditelosomics of CS with the 4 sets of primers, indicating that the major restoring gene in LK783 was located on IBS, but the relative location of the gene was different from Rfv1, allelism of the two genes should be further investigated. The breeding for new fertility restorer lines of K-type cytoplasmic male sterility in wheat would be facilitated by using the four polymorphic markers.

Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

Somatic hybridization is performed to obtain significant cytoplasmic male sterility （CMS） lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-IA, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-IB, and the abortive process of SaNa-IA were described through phenotypic observations and microtome sections. The floral organ of the sterile line SaNa-IA was sterile with a shortened filament and deflated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-IA aborted in the pollen mother cell （PMC） stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-IA is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

Development of Japonica Male Sterile Lines Integrating Cytoplasmic Male Sterility and Photosensitive Genic Male Sterility

Acknowledgement It has been previously established that the BT type of cytoplasmic male sterility （CMS） is induced by high temperatures, while photosensitive genic male sterility （PGMS） seed sets by low temperatures induce. In the current study, we have bred photosensitive cytoplasmic male sterility （PCMS） lines （2308SA and 2310SA） by crossing the CMS line with the PGMS japonica line with maintainer genes. The sterility of PCMS japonica was consequently controlled by two groups of male sterile genes resulting from the integration of PGMS and CMS genes. The results on plant fertility, at different sowing times, were as follows： （a） Under conditions of natural long-day photoperiod and at temperatures above 35~C, the PGMS gene regulated PCMS japonica sterility - the higher the temperature, the lower the pollen fertility. However, bagged seed sets of PCMS japonica, not exposed to high temperatures, induced the CMS seed set. （b） Exposure to long-day photoperiod and temperature conditions between 35℃ and the critical sterility inducing temperature of PGMS resulted in both PGMS and CMS gene controlled sterility of PCMS japonica, which exhibited stable characteristics. （c） When exposed to critical sterility inducing temperatures or short-day photoperiod and daily high temperatures below 32℃, the BT type of the CMS gene regulated PCMS sterility. Under these conditions, the PGMS gene rendered male sterility insusceptible to occasional cool summer days when this PCMS line, adopted for hybrid seed production, develops into panicle differentiation stage. The present study also investigated the fertility restoration, seed production and combining ability of PCMS japonica so as to optimize its use.

Mitochondrial Proteomic Analysis of Cytoplasmic Male Sterility Line and Its Maintainer in Wheat (Triticum aestivum L.)

To investigate the CMS mechanism of wheat on proteomic level and find the crucial proteins which related to fertility,mitochondria was isolated from young spike of wheat by differential centrifugation and Percoll density-gradient methods.Determined by marker enzyme assays and chlorophyll content,the mainly contaminants in the spike mitochondrial fraction were caused by peroxisomes,plastids and chloroplasts after the first discontinuous Percoll density gradient centrifugation.In order to improve the purity of spike mitochondria,a second 28% Percoll self-forming density gradient centrifugation was further carried out,the result showed that the contaminants were decreased to negligible amount,meanwhile the integrity of mitochondria （88%） was improved to 90%.The spike mitochondria proteins extracted from uninucleate stage of （S）-1376A and （A）-1376B were separated by two-dimensional electrophoresis （2-DE）,and the silver stained gels were analyzed by PDQuest 2-DE software,about 326 protein spots could be visualized on the 2-DE maps,and also revealed a similar pattern between the male sterile line and its maintainer line,except 11 spots were differentially expressed.A total of five differentially expressed proteins were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS）,three of them were identified as manganese superoxide dismutase and T5E216 following NCBInr database by the Mascot software.These results may contribute to further understanding of the mechanisms of CMS in wheat.

Establishment and Identification of Cytoplasmic Male Sterility in Brassica napus by Intergeneric Somatic Hybridization

Exploitation of novel cytoplasmic male sterility (CMS) is a main approach for widening the cytoplasmic genetic background of hybrid oilseed rape and avoiding epidemic risk in oilseed rape production. In this study, symmetric somatic hybrids between Brassica napus var. Zhongshuang4 and Sinapis arvensis (Ye- you18) were produced by protoplast fusion. Two of the six established hybrids were male sterile showing trace or no pollen release upon flowering with non- or slightly extended stamens. Using Zhongshuang4 as a recurrent parent to pollinate the male sterile plants, the ratio of male sterile plants increased with the number of backcrosses. As early as in BC 3 generation, most of the sterile families had nearly 100% sterile plants. Up to BC 4 generation, the male sterility became stable and no fertility segregation was observed. All F 1 progenies from tested crosses using restorer and maintainer lines of Polima CMS were 100% sterile, indicating that the established CMS by somatic hybridization is different from Polima CMS. The origin of the cytoplasm and potential use of this novel CMS in oilseed rape breeding were discussed.

Nectar secretion of RN-type cytoplasmic male sterility three lines in soybean [Glycine max(L.) Merr.]

Significant progress has been achieved in the use of heterosis in soybean and several soybean hybrids have been released in China. However, broad use of hybrid soybean seed is limited due to low seed setting of female parents. Breeding cytoplasmic male sterile（CMS） lines with high out-crossing rate is necessary to solve the problem. The objective of this study was to determine the relationship between out-crossing rate of CMS lines and their nectar secretion. The daily nectar secretion rhythm, meteorological effect on nectar secretion, and differences in nectar secretion among genotypes and years were investigated in 27 soybean CMS lines（A-lines） with their maintainers（B-lines） and restorers（R-lines）. The correlation between out-crossing rate of CMS lines and nectar production was also evaluated. Nectar secretion had diurnal variation. Secretion initiated at about 06：00 for most materials and reached a peak at 07：00–08：30 after flower opened, then the nectar secretion decreased gradually. A sub-peak appeared at about 13：00, while the nectar could not be detected at 17：00. Nectar secretion was greatly influenced by the weather conditions. The amount of nectar secretion increased gradually over time during periods of high temperature and no rainfall for several days. Rainy weather and low temperatures inhibited nectar secretion. There were obvious variations of nectar amount among different genotypes tested. Significant nectar variation within a genotype among years was also observed, and the highest nectar secretion was 3-fold higher than the lowest. The amount of nectar secretion from R-lines was significantly higher than that of A-and B-lines. There was no significant difference in nectar secretion between A-and B-lines. A-and B-lines with higher out-crossing rates secreted more nectar. The amount of nectar secretion of A-and B-lines were significantly positively correlated with the out-crossing rate of A-lines.

Mapping of Rice Fertility-Restoring Genes for ID-Type Cytoplasmic Male Sterility in a Restorer Line R68

An F2 population derived from the cross Zhong 9A/R68 was used to map the fertility-restoring （Rf） gene for ID-type cytoplasmic male sterility （CMS）. Two bulks （a fertile bulk and a sterile bulk） were constructed by pooling equal amount of ten highly fertile lines and ten highly sterile lines, respectively. Four hundred and thirteen pairs of simple sequence repeat （SSR） primers, which evenly distributed on 12 chromosomes of rice, were selected for analyzing polymorphisms between the parents and between the two bulks. The primer RM283 on chromosome 1 and the primers RM5756, RM258, RM6100 and RM171 on chromosome 10 were found to be polymorphic between the parents and between the two bulks. These five SSR markers were linked to fertility-restoring genes. A total of 82 excessive sterile lines were selected from Zhong 9A/R68 F2 population to estimate the genetic distance between five SSR markers and fertility-restoring genes respectively. The results indicated that one Rf gene was linked to RM283 located on chromosome 1 at a distance of 6.7 cM, and the other Rfgene was mapped to the long arm of chromosome 10 flanked by RM258 and RM6100 at the distances of 8.0 cM and 2.4 cM, respectively.

Cytoplasmic Male Sterility in Maize

14 isoplasmic and allonuclear cytoplasmic male sterile lines were used as female parents, 8 tester lines as male parents, 101 F, progenies were obtained. Fertility restoration response of 101 F] progenies were investigated through field observation and pollen stainability examination under microscope. 14 isoplasmic and allonuclear cytoplasmic male sterile lines were developed by repeated backcross with recurrent male parent lines for more than 8 generations. The result shows: tester line Zifengl not only restored the isoplasmic and allonuclear sterile lines of group C backcrossed with Mo17, Yu30 and Heer, but also completely restored the isoplasmic and allonuclear cytoplasm male sterile lines of group T backcrossed with Mo17, HZS , 1792 , 292 and Yu30. Therefore, nuclear background limits the use of Zifengl as a tester for identification of cytoplasmic male sterility. Furthermore RFLPs of mitochondrial DNA of 6 isonuclear and alloplasmic cytoplasmic male sterile lines were analyzed with BamH I and Hind 1 restriction endonuclease and mitochondrial DNA probes pBcmH3 and Cox II . The same RFLPs were found within sterile cytoplasm of group C, including C, Chuan G, Lei 2 and Lei 3, but a different RFLP pattern was observed among sterile cytoplasm of group S, C, T and the normal cytoplasm. This result suggested that the RFLP markers tightly linked to sterile mitochon-drial genes of different groups could be applied in the identifcation of cytoplasmic male sterility.

Application of hybrids with cytoplasmic male-sterility in Zea mays L.in China

The abortive behaviour and the cytoplasm quality of cytoplasmic male-sterility(CMS)lines of homocaryon were investigated and the existence of C race of Bipolaris maydiswere discussed from the point of view of genetic breeding,physiology and pathology in this paper.Then the countermeasures to prevent the danger from disease of B. maydis in CMS hybrid pro-duction were proposed.

Inheritance of Fertility Restoration for Cytoplasmic Male Sterility in a New Gossypium barbadense Restorer

In order to clarify inheritance mechanism of fertility restoration for cytoplasmic male sterility （CMS） in a new Gossypium barbadense restorer line Hai R which was found in the fertility test crossing of G. hirsutum CMS lines with G. barbadense germplasms. 23 fertility segregation populations of F2 and backcross were used to analyze the inheritance of fertility restoring gene（s） of Hai R. The result showed that Hai R had one major dominant gene （RfB） to control the CMS fertility restoration and this fertility restoration gene functioned at the sporophytic level. The sterile cytoplasm background might not only influence the transmission rate of male gamete but also that of female gamete when the restorer gene was recessive. It could be deduced that this fertility restoration gene might come from G. harknessii cotton, Hai R is of value in the application of cotton interspecific hybrid breeding.

Development of Transgenic Restorer of Cytoplasmic Male Sterility in Upland Cotton

A glutathione S-transferase gene (gst) has been introduced into restorers of cytoplasmic male sterility in upland cotton (Gossypium hirsutum L.) using Agrobacterium-mediated transformation. A trans-genie restorer, signed as 'Zheda strong restorer', which has strong restorability to male sterility, was selected from progeny plants of transformants. When compared with an American restorer 'DES-HAF277', the fertility restorability of 'Zheda strong restorer' to male sterility was been enhanced by 25.8% in the percentage of viable pollens of hybrid (sterile line × restorer) F1 The hybrid gave 3.6 more bolls per plant, 10.1% less aborted seeds and 10.6% more lint yield when 'Zheda strong restorer' was used as male parent than when 'DES-HAF277' was. Southern and Northern bloting analysis showed that the foreign gsf gene was detectable and highly expressed in 'Zheda strong restorer'.

棉花CMS-D2和CMS-D8不育系线粒体全基因组比较分析

【目的】探究哈克尼西棉不育胞质(CMS-D2)和三裂棉不育胞质(CMS-D8)的不育系线粒体基因组之间的序列结构差异,为筛选鉴定不育相关基因奠定基础。【方法】根据D2A和D8A这2个不育系的线粒体基因组测序组装结果,使用Synteny and Rearrangement Identifier(Sy RI)软件鉴定结构变异,用Plotsr可视化分析包含共线性及非共线性区域的重组变异位点。以D8A线粒体基因组注释结果为参考,用D2A线粒体基因组注释的开放阅读框(open reading frame,ORF)编码的氨基酸序列进行tblastn比对,筛选出D2A线粒体基因组中特有的ORF,并进行聚合酶链式反应(polymerase chain reaction,PCR)验证、相对表达量分析和生物信息学分析。【结果】D2A和D8A这2个不育系线粒体基因组之间存在2个部分重叠且相邻的倒易位区域。在D2A中发现17个特异的ORF,PCR验证了D2A中存在的6个特异ORF(orf114e、orf121b-1、orf121b-2、orf138b-2、orf186a-2和orf317a-2)。3~4 mm花蕾中orf121b-1和orf121b-2的相对表达量较高。orf114e、orf186a-2和orf317a-2具有典型的跨膜结构域和嵌合基因结构,符合不育基因的部分特征。【结论】D2A和D8A线粒体基因组间存在2个相邻的倒易位区域。D2A中存在6个特异的ORF,其中orf114e、orf186a-2和orf317a-2可能与棉花CMS-D2孢子体败育有关。

The unstability of cytoplasmic male sterility of Brassica napus L.

The unstability of cytoplasmic male sterilities (CMS) of Brassica napus Lwas stud-ied from 1989 to 1992,and the results indicated: 1. The occurrence of trace-pollen plants In sterilematerials was caused by nuclear polygenes of the maintainers; 2. Progenies of partial sterility typeshowed segregation of sterile, partially sterile (with trace pollen )and fertile plants; 3. High andlow temperature CMS lines were crossed and its progenies manifested complex segregation; 4.There was a tendency to increase the fertility with successive selfing of the high temperatureCMS line.

Influential Factors of Restorer of New Cytoplasmic Male Sterile(NER) on Anther Culture

Some influential factors of anther culture were studied preliminarily by conducting anther culture of the restorers of new cytoplasmic male sterile （NER）. Several results were obtain from this experiment and they were listed as follow：① MS cultrure medium with such hormones as 2,4-D 2 mg/L,6-BA 0.5 mg/L, NAA 0.5 mg/L was the best suitable for callus induction of NER. ②The difference of induction rate was significantly different between different plant age groups. From the 110th day to 141th day,the induction rate was increased with the increase of age and the difference of induction rate reached 0.01 significant difference level. The induction rate reached the highest value in the 141th day then it declined gradually. ③The combined use of 2, 4-D and 6-BA with proper increase of 2,4-D was good for inducing callus. ④The green plantlet induction rate of NER was increased when the concentration of 6-BA increased from 2 mg/L to 4 mg/L. Adding ZT from 0.5 mg/L to 2 mg/L. 6-BA would led 2.47% increase of green plantlet olantlet induction rate.

Development of New InDel Marker to Detect Genotypes of Rf-1a Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility in Rice

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id^ntifio.~tinn ~nd m^rkp.r-~.~.~i^fp.d .~.tAr.tinn （MA.~ in hr~=dinn i^nnnir~ r^fnr~=r lin==~